The present invention relates to a novel composition for permeabilizing microorganism walls and to the uses thereof, in particular the use thereof in a method for counting and/or for the targeted identification of the microorganisms in a liquid or gaseous medium.
The invention applies to the control of the microbiological quality of the liquid and gaseous media involved in production lines for food or pharmaceutical products.
In this field, the microorganisms to be detected are often present in very small number and it is advisable to distinguish the nature of the microbes in order to determine whether they present a risk to human health. The control carried out must warn of any contamination of the manufactured product within a short period of time, making it possible, where appropriate, to stop the manufacture thereof and to take decontamination steps.
Many methods have been developed in order to minimize the culturing time for microorganisms, while at the same time making it possible to search for certain microbes.
This is the subject in particular of the methods for detecting microorganisms carried out after filtration of the liquid through a membrane, described in application WO 01/59157. According to this method, the microorganisms contained in a liquid sample are retained at the surface of a membrane by passing the liquid through said membrane. The microorganisms are cultured at the surface of the membrane in contact with an agar culture medium for the amount of time necessary to form a microcolony not visible to the naked eye. The cells forming the microcolony are then lyzed in order to release their adenosine triphosphate (ATP) and nucleic acid content. The ATP is used as a marker for identifying and quantifying the live cells by ATP-bioluminescence. The detection is termed universal in that ATP is a marker that is present in all living microorganisms. Reading of the result is obtained by means of a chemiluminescence reaction involving the conversion of the ATP into photons by an enzyme specific for said ATP. The light signal is detected using an appropriate video interface (e.g.: LCD camera). The image obtained makes it possible to visualize in situ the site on the membrane where the microbe has developed, in a manner similar to conventional counting carried out on a Petri dish in agar medium.
The “Milliflex Rapid®” system, sold by the company Millipore, operates on the principle described above. It was designed in order to carry out the filtration and detection steps on one and the same membrane, which is maintained on a plastic support. This support is designed so as to fit onto the various devices that are used for the filtration, the culturing of the microorganisms, the impregnation of the membrane with detection reagents and the taking of images in a video chamber.
This miniaturized system allows an appreciable gain in time compared with the conventional tests on Petri dishes. Furthermore, it makes it possible to store the images obtained on a digital support, which makes it possible to monitor the contaminations (traceability).
However, this system has certain limits due to the fact that, in practice, the counting of the microorganisms present at the surface of the membrane is carried out after cell lysis. It is, consequently, difficult to reuse the same membrane with a view to accurately identifying the microorganisms detected.
However, in order to demonstrate the presence of certain microorganisms, it is possible to carry out a specific hybridization using labeled probes, whether they are nucleotide in nature or else of PNA (peptide nucleic acid) type.
PNA probes have the advantage of being peptide in nature, which makes it possible, in certain applications, to advantageously replace oligonucleotide probes.
In general, in situ probe hybridization requires complete accessibility to the nucleic acids of the cell otherwise this manipulation generates false negatives.
Application WO 2004/050902 describes a system of detection on a membrane which makes it possible to detect the presence of microorganisms contaminating blood samples.
The particularity of this system lies in the fact that the microorganisms are detected through the penetration of labeling agents into the microorganisms through their wall. The labeling agents used are small molecules, in particular compounds which intercalate nucleic acids (DNA, RNA), such as cyanin derivatives, propidium iodide, acridine orange or ethidium bromide, which poses less difficulty in terms of penetration into the microorganisms than oligonucleotide probes.
The liquid sample to be tested is diluted in a permeabilization solution comprising the labeling agent and a cell permeabilization reagent. The cells are incubated in this permeabilization solution, the aim of which is to increase the cell wall porosity, so as to facilitate the penetration of the labeling product into the microorganisms present. The permeabilization solution is subsequently filtered, with the possible microorganisms that it contains, through a membrane. The microorganisms present are then retained on the membrane, and are then detected by reacting the labeling agent with a compound and/or a light source which allows the emission of a fluorescent signal. This system has the advantage of not lyzing the microorganisms, or at least of limiting as much as possible their destruction before their detection.
This results in a detection that offers better resolution.
However, because the markers are not specific, this system does not make it possible to determine the nature of the microorganisms detected.
Furthermore, in such a system, the handling of the microorganisms takes place in solution, which requires specific precautions and equipment which is more complex. It should be noted that the incubation of the live cells in solution before filtration has the drawback that the cells can continue to grow and to divide before being fixed on the membrane, which casts some uncertainty on the final result of the test.
It is also difficult to suitably meter out the penetration reagent into a permeabilization solution according to the method described in WO 2004/050902, in particular when it is sought to simultaneously detect gram-positive bacteria, which are generally more sensitive to permeabilizing agents, and more resistant gram-negative bacteria.
The permeabilization agents which may go to make up the composition of the permeabilization solution are typically ethylenediaminetetraacetic acid (EDTA), polyethylene glycol (PEG), digitonin, nonensin, sodium hexamethaphosphate or benzalkonium chloride.
It is also possible to use polyethyleneimine (PEI), which is a slightly basic and polycationic aliphatic polymer known to make bacterial walls permeable to solutes, such as antibiotics, which do not normally penetrate into the cytoplasma bacteria [Helander, I. M. et al., Polyethyleneimine is an effective permeabilizer of gram-negative bacteria, Microbiology (1997), 143: 3193-3199].
The concentration of polyethyleneimine in the permeabilization solutions such as that described in WO 2004/050902 is limited to concentrations of generally less than 100 μg/ml. The concentration most suitable for gram-negative bacteria, such as Esherichia coli, is around 20-30 μg/ml.